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1.
Tokai J Exp Clin Med ; 45(3): 148-151, 2020 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-32901905

RESUMO

PURPOSE: Pheochromocytoma (PCC) and paraganglioma (PGL) associated with the succinate dehydrogenase (SDH) germline mutations are characterized by negative results of immunohistochemistry tests for SDH subunit B (SDHB). Genetic testing for the SDH complex (SDHA, SDHB, SDHC, SDHD, and SDHAF2) is indicated only in patients with those diseases in whom immunohistochemistry tests for SDHB as a surrogate marker to detect the SDH complex mutation yield negative results. Two novel SDHB germline mutations, L157X and P236S, in PGL were previously reported. We therefore examined immunohistochemistry testing for SDHB in the PGLs with the SDHB germline mutations of L157X and P236S. METHODS: Immunohistochemistry for SDHB was performed in PGLs with the SDHB germline mutations of L157X and P236S. Five cases of sporadic PCC were subject to immunohistochemistry testing for SDHB. Normal tissue from the adrenal cortex adjacent to the sporadic PCC was used as the external positive control. RESULTS: Immunohistochemistry results were positive for SDHB in PGLs with the SDHB germline mutation of L157X and P236S, all five cases of sporadic PCC, and the adrenal cortex as the external positive control. CONCLUSION: Immunohistochemistry tests for SDHB showed positivity in PGLs associated with the SDHB germline mutations of L157X and P236S. Thus, immunohistochemistry testing for SDHB might not always reveal a surrogate marker in formal genetic testing of the SDH complex.


Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Biomarcadores Tumorais/metabolismo , Mutação em Linhagem Germinativa , Paraganglioma/genética , Coloração e Rotulagem/métodos , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Córtex Suprarrenal/metabolismo , Humanos , Imuno-Histoquímica , Resultados Negativos
2.
Croat Med J ; 61(4): 354-365, 2020 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-32881434

RESUMO

AIM: To analyze axon morphology on rapid Golgi impregnated pyramidal neurons in the dorsolateral prefrontal cortex in schizophrenia. METHODS: Postmortem brain tissue from five subjects diagnosed with schizophrenia and five control subjects without neuropathological findings was processed with the rapid Golgi method. Layer III and layer V pyramidal neurons from Brodmann area 9 were chosen in each brain for reconstruction with Neurolucida software. The axons and cell bodies of 136 neurons from subjects with schizophrenia and of 165 neurons from control subjects were traced. The data obtained by quantitative analysis were compared between the schizophrenia and control group with the t test. RESULTS: Axon impregnation length was consistently greater in the schizophrenia group. The axon main trunk length was significantly greater in the schizophrenia than in the control group (93.7 ± 36.6 µm vs 49.8 ± 9.9 µm, P = 0.032). Furthermore, in the schizophrenia group more axons had visibly stained collaterals (14.7% vs 5.5%). CONCLUSION: Axon rapid Golgi impregnation stops at the beginning of the myelin sheath. The increased axonal staining in the schizophrenia group could, therefore, be explained by reduced axon myelination. Such a decrease in axon myelination is in line with both the disconnection hypothesis and the two-hit model of schizophrenia as a neurodevelopmental disease. Our results support that the cortical circuitry disorganization in schizophrenia might be caused by functional alterations of two major classes of principal neurons due to altered oligodendrocyte development.


Assuntos
Axônios/patologia , Córtex Pré-Frontal/patologia , Células Piramidais/patologia , Esquizofrenia/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Complexo de Golgi/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Coloração e Rotulagem/métodos
3.
PLoS One ; 15(8): e0237748, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866195

RESUMO

Soil microbiota are considered a source of undiscovered bioactive compounds, yet cultivation of most bacteria within a sample remains generally unsuccessful. Two main reasons behind the unculturability of bacteria are the presence of cells in a viable but not culturable state (such as dormant cells) and the failure to provide the necessary growth requirements in vitro (leading to the classification of some bacterial taxa as yet-to-be-cultured). The present work focuses on the development of a single procedure that helps distinguish between both phenomena of unculturability based on viability staining coupled with flow cytometry and fluorescence-activated cell sorting. In the selected soil sample, the success rate of cultured bacteria was doubled by selecting viable and metabolically active bacteria. It was determined that most of the uncultured fraction was not dormant or dead but likely required different growth conditions. It was also determined that the staining process introduced changes in the taxonomic composition of the outgrown bacterial biomass, which should be considered for further developments. This research shows the potential of flow cytometry and fluorescence-activated cell sorting applied to soil samples to improve the success rate of bacterial cultivation by estimating the proportion of dormant and yet-to-be-cultured bacteria and by directly excluding dormant cells from being inoculated into growth media.


Assuntos
Bactérias/crescimento & desenvolvimento , Técnicas Microbiológicas/métodos , Microbiota/fisiologia , Microbiologia do Solo , Bactérias/química , Bactérias/genética , Biomassa , Separação Celular/métodos , Meios de Cultura , DNA Bacteriano/isolamento & purificação , Estudos de Viabilidade , Citometria de Fluxo/métodos , Corantes Fluorescentes/química , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Coloração e Rotulagem/métodos
4.
Nat Commun ; 11(1): 4339, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859909

RESUMO

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) facilitates multiplexing in superresolution microscopy but is practically limited by slow imaging speed. To address this issue, we propose the additions of ethylene carbonate (EC) to the imaging buffer, sequence repeats to the docking strand, and a spacer between the docking strand and the affinity agent. Collectively termed DNA-PAINT-ERS (E = EC, R = Repeating sequence, and S = Spacer), these strategies can be easily integrated into current DNA-PAINT workflows for both accelerated imaging speed and improved image quality through optimized DNA hybridization kinetics and efficiency. We demonstrate the general applicability of DNA-PAINT-ERS for fast, multiplexed superresolution imaging using previously validated oligonucleotide constructs with slight modifications.


Assuntos
Técnicas Citológicas/métodos , DNA/química , Microscopia de Fluorescência/métodos , Simulação de Acoplamento Molecular/métodos , Linhagem Celular , Humanos , Processamento de Imagem Assistida por Computador/métodos , Oligonucleotídeos , Coloração e Rotulagem/métodos
5.
Nat Commun ; 11(1): 3850, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737322

RESUMO

Resolving the distribution of specific proteins at the nanoscale in the ultrastructural context of the cell is a major challenge in fluorescence microscopy. We report the discovery of a new principle for an optical contrast equivalent to electron microscopy (EM) which reveals the ultrastructural context of the cells with a conventional confocal microscope. By decrowding the intracellular space through 13 to 21-fold physical expansion while simultaneously retaining the proteins, bulk (pan) labeling of the proteome resolves local protein densities and reveals the cellular nanoarchitecture by standard light microscopy.


Assuntos
Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Proteoma/análise , Coloração e Rotulagem/métodos , Acrilamidas/química , Reagentes para Ligações Cruzadas/química , Corantes Fluorescentes/química , Células HeLa , Humanos , Hidrogéis/química , Espaço Intracelular/química , Succinimidas/química , Inclusão do Tecido/métodos
6.
Rinsho Shinkeigaku ; 60(8): 565-568, 2020 Aug 07.
Artigo em Japonês | MEDLINE | ID: mdl-32641630

RESUMO

A 49-year-old woman was admitted to our hospital with suspected hypertensive encephalopathy. On the basis of MRI showing leptomeningeal enhancement and Class V cytology of the CSF, she was diagnosed as having leptomeningeal carcinomatosis. Although no primary site was detected, a few melanin granules were observed at the third CSF examination. The atypical cells in the CSF demonstrated immunoreactivity for HMB-45 and S-100, which are specific markers of malignant melanoma. There have been few reports of meningeal melanomatosis in Japan. This case illustrates that immunostaining is diagnostically useful in patients with leptomeningeal carcinomatosis from neoplasms with unknown primary sites.


Assuntos
Biomarcadores Tumorais/líquido cefalorraquidiano , Líquido Cefalorraquidiano , Citodiagnóstico/métodos , Melanoma/líquido cefalorraquidiano , Melanoma/diagnóstico , Carcinomatose Meníngea/líquido cefalorraquidiano , Carcinomatose Meníngea/diagnóstico , Neoplasias Meníngeas/líquido cefalorraquidiano , Neoplasias Meníngeas/diagnóstico , Coloração e Rotulagem/métodos , Idoso , Feminino , Humanos , Antígenos Específicos de Melanoma/líquido cefalorraquidiano , Neoplasias Primárias Desconhecidas , Proteínas S100/líquido cefalorraquidiano
7.
Nat Commun ; 11(1): 3699, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709877

RESUMO

Mitochondria play a critical role in generating energy to support the entire lifecycle of biological cells, yet it is still unclear how their morphological structures evolve to regulate their functionality. Conventional fluorescence microscopy can only provide ~300 nm resolution, which is insufficient to visualize mitochondrial cristae. Here, we developed an enhanced squaraine variant dye (MitoESq-635) to study the dynamic structures of mitochondrial cristae in live cells with a superresolution technique. The low saturation intensity and high photostability of MitoESq-635 make it ideal for long-term, high-resolution (stimulated emission depletion) STED nanoscopy. We performed time-lapse imaging of the mitochondrial inner membrane over 50 min (3.9 s per frame, with 71.5 s dark recovery) in living HeLa cells with a resolution of 35.2 nm. The forms of the cristae during mitochondrial fusion and fission can be clearly observed. Our study demonstrates the emerging capability of optical STED nanoscopy to investigate intracellular physiological processes with nanoscale resolution for an extended period of time.


Assuntos
Ciclobutanos , Membranas Mitocondriais/ultraestrutura , Nanotecnologia/métodos , Fenóis , Linhagem Celular , Corantes Fluorescentes/química , Células HeLa , Humanos , Microscopia de Fluorescência/métodos , Mitocôndrias , Dinâmica Mitocondrial/fisiologia , Coloração e Rotulagem/métodos
8.
Nat Commun ; 11(1): 3284, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32601292

RESUMO

The inner nuclear membrane (INM) selectively accumulates proteins that are essential for nuclear functions; however, overaccumulation of INM proteins results in a range of rare genetic disorders. So far, little is known about how defective, mislocalized, or abnormally accumulated membrane proteins are actively removed from the INM, especially in plants and animals. Here, via analysis of a proximity-labeling proteomic profile of INM-associated proteins in Arabidopsis, we identify critical components for an INM protein degradation pathway. We show that this pathway relies on the CDC48 complex for INM protein extraction and 26S proteasome for subsequent protein degradation. Moreover, we show that CDC48 at the INM may be regulated by a subgroup of PUX proteins, which determine the substrate specificity or affect the ATPase activity of CDC48. These PUX proteins specifically associate with the nucleoskeleton underneath the INM and physically interact with CDC48 proteins to negatively regulate INM protein degradation in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Membrana Nuclear/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plantas Geneticamente Modificadas , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Proteoma/genética , Coloração e Rotulagem/métodos , Espectrometria de Massas em Tandem/métodos , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
9.
J Surg Oncol ; 122(2): 226-233, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32436255

RESUMO

BACKGROUND AND OBJECTIVES: Recently, PINPOINT, a novel laparoscopic fusion indocyanine green fluorescence imaging (IGFI) system has become available for laparoscopic liver resection. This study aims to characterize fluorescence patterns of intrahepatic cholangiocarcinoma (ICC) using the negative counterstaining method in laparoscopic anatomical hepatectomies of ICC. METHODS: Eleven consecutive patients, diagnosed with intrahepatic cholangiocarcinoma and underwent laparoscopic liver resection between April 2017 and December 2018, were retrospectively reviewed. A laparoscopic IGFI navigation system was used to characterize fluorescence patterns of ICC with intraoperative liver segment demarcation by means of negative counterstaining. RESULTS: Fusion IGFI of ICC was successfully obtained from all 11 patients from the surgical specimens. The fluorescence patterns of ICC can be categorized into rim-type fluorescence and segmental fluorescence, depending on tumor growth. In eight patients, indocyanine green fluorescence imaging was used to identify the hepatic lobes or segments by negative counterstaining. In six cases, a valid and persistent demarcation was achieved intraoperatively. CONCLUSION: Laparoscopic IGFI system could identify different types of ICC lesions and may facilitate real-time navigation for laparoscopic anatomic liver resection of ICC.


Assuntos
Neoplasias dos Ductos Biliares/diagnóstico por imagem , Neoplasias dos Ductos Biliares/cirurgia , Colangiocarcinoma/diagnóstico por imagem , Colangiocarcinoma/cirurgia , Verde de Indocianina/administração & dosagem , Imagem Óptica/métodos , Idoso , Idoso de 80 Anos ou mais , Corantes/administração & dosagem , Estudos de Viabilidade , Feminino , Humanos , Período Intraoperatório , Laparoscopia , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Cuidados Pré-Operatórios/métodos , Estudos Retrospectivos , Coloração e Rotulagem/métodos
10.
Neuron ; 106(3): 369-387, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32380050

RESUMO

Tissue clearing and light-sheet microscopy have a 100-year-plus history, yet these fields have been combined only recently to facilitate novel experiments and measurements in neuroscience. Since tissue-clearing methods were first combined with modernized light-sheet microscopy a decade ago, the performance of both technologies has rapidly improved, broadening their applications. Here, we review the state of the art of tissue-clearing methods and light-sheet microscopy and discuss applications of these techniques in profiling cells and circuits in mice. We examine outstanding challenges and future opportunities for expanding these techniques to achieve brain-wide profiling of cells and circuits in primates and humans. Such integration will help provide a systems-level understanding of the physiology and pathology of our central nervous system.


Assuntos
Encéfalo/citologia , Imageamento Tridimensional/métodos , Imagem Óptica/métodos , Coloração e Rotulagem/métodos , Animais , Encéfalo/fisiologia , Humanos , Microscopia/métodos
11.
World J Microbiol Biotechnol ; 36(5): 76, 2020 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-32390085

RESUMO

The detection and monitoring of Vibrio parahaemolyticus pathogen in aquatic foods have become essential for preventing outbreaks. In this study, loop-mediated isothermal amplification (LAMP) assay with the azo dye, hydroxynaphthol blue (HNB) was developed targeting species-specific tlh gene. The assay was carried out on 62 seafood samples that included clam and shrimp and compared with conventional LAMP assay performed with the commonly used fluorescent dye, conventional PCR, and real-time PCR (RT-PCR). Of 62 samples studied for tlh gene, 32 (51.61%) gave positive by HNB-LAMP, which comprised 22 (70.96%) clam samples and 10 (32.25%) shrimp samples. The HNB-LAMP assay was found to be highly sensitive, specific, and superior to conventional PCR (p > 0.05). RT-PCR presented higher sensitivity than HNB-LAMP; however, it has the limitation of being cost-intensive and requiring technical expertise to perform. HNB-LAMP is affordable, rapid, simple, and easy to perform, allowing naked eye visualization.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Naftalenossulfonatos , Técnicas de Amplificação de Ácido Nucleico/métodos , Alimentos Marinhos/microbiologia , Vibrio parahaemolyticus/isolamento & purificação , Corantes , Microbiologia de Alimentos/métodos , Naftalenossulfonatos/química , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos , Raios Ultravioleta
12.
Nat Methods ; 17(6): 609-613, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32424271

RESUMO

We developed entangled link-augmented stretchable tissue-hydrogel (ELAST), a technology that transforms tissues into elastic hydrogels to enhance macromolecular accessibility and mechanical stability simultaneously. ELASTicized tissues are highly stretchable and compressible, which enables reversible shape transformation and faster delivery of probes into intact tissue specimens via mechanical thinning. This universal platform may facilitate rapid and scalable molecular phenotyping of large-scale biological systems, such as human organs.


Assuntos
Hidrogéis/química , Coloração e Rotulagem/métodos , Engenharia Tecidual/métodos , Acrilamida/química , Animais , Fenômenos Biomecânicos , Materiais Biomiméticos/química , Bioimpressão , Córtex Cerebral/química , Reagentes para Ligações Cruzadas/química , Módulo de Elasticidade , Hipocampo/química , Humanos , Teste de Materiais , Camundongos , Estresse Mecânico , Resistência à Tração
13.
Diagn Cytopathol ; 48(8): 813-815, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32396261

RESUMO

The COVID-19 pandemic is posing a worldwide challenge to control and contain. SARS-CoV-2 is a highly infectious virus. Health care providers at the front lines are at high risk of getting the infection and the risk applies also to laboratory personnel as they deal with specimens that might be contaminated with infectious materiel. Cytopathology teams specifically are at high risk of dealing with contaminated material because of patients encounter during fine-needle aspiration biopsies or Rapid On-Site Evaluation (ROSE) for adequacy. In our article, we discuss alternative safer staining methods to the widely used Diff-Quick stain that can be utilized for ROSE to decrease the risk of viral exposure during the current COVID-19 pandemic.


Assuntos
Betacoronavirus , Infecções por Coronavirus/transmissão , Pessoal de Laboratório Médico , Saúde do Trabalhador , Pandemias , Pneumonia Viral/transmissão , Coloração e Rotulagem/métodos , Biópsia por Agulha Fina , Corantes , Infecções por Coronavirus/prevenção & controle , Humanos , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle
14.
Parasit Vectors ; 13(1): 192, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32293537

RESUMO

BACKGROUND: Prior to a major release campaign of sterile insects, including the sterile insect technique, male mosquitoes must be marked and released (small scale) to determine key parameters including wild population abundance, dispersal and survival. Marking insects has been routinely carried out for over 100 years; however, there is no gold standard regarding the marking of specific disease-transmitting mosquitoes including Anopheles arabiensis, Aedes aegypti and Aedes albopictus. The research presented offers a novel dusting technique and optimal dust colour and quantities, suitable for small-scale releases, such as mark-release-recapture studies. METHODS: We sought to establish a suitable dust colour and quantity for batches of 100 male An. arabiensis, that was visible both by eye and under UV light, long-lasting and did not negatively impact longevity. A set of lower dust weights were selected to conduct longevity experiments with both Ae. aegypti and Ae. albopictus to underpin the optimal dust weight. A further study assessed the potential of marked male An. arabiensis to transfer their mark to undusted males and females. RESULTS: The longevity of male An. arabiensis marked with various dust colours was not significantly reduced when compared to unmarked controls. Furthermore, the chosen dust quantity (5 mg) did not negatively impact longevity (P = 0.717) and provided a long-lasting mark. Dust transfer was found to occur from marked An. arabiensis males to unmarked males and females when left in close proximity. However, this was only noticeable when examining individuals under a stereomicroscope and thus deemed negligible. Overall, male Ae. aegypti and Ae. albopictus displayed a greater sensitivity to dusting. Only the lowest dust weight (0.5 mg) did not significantly reduce longevity (P = 0.888) in Ae. aegypti, whilst the lowest two dust weights (0.5 and 0.75 mg) had no significant impact on longevity (P = 0.951 and 0.166, respectively) in Ae. albopictus. CONCLUSION: We have devised a fast, inexpensive and simple marking method and provided recommended dust quantities for several major species of disease-causing mosquitoes. The novel technique provides an evenly distributed, long-lasting mark which is non-detrimental. Our results will be useful for future MRR studies, prior to a major release campaign.


Assuntos
Aedes , Anopheles , Coloração e Rotulagem , Animais , Poeira , Corantes Fluorescentes , Infertilidade Masculina , Longevidade , Masculino , Controle de Mosquitos/métodos , Coloração e Rotulagem/métodos
16.
Proc Natl Acad Sci U S A ; 117(17): 9223-9231, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32284403

RESUMO

Many medical and biological protocols for analyzing individual biological cells involve morphological evaluation based on cell staining, designed to enhance imaging contrast and enable clinicians and biologists to differentiate between various cell organelles. However, cell staining is not always allowed in certain medical procedures. In other cases, staining may be time-consuming or expensive to implement. Staining protocols may be operator-sensitive, and hence may lead to varying analytical results, as well as cause artificial imaging artifacts or false heterogeneity. We present a deep-learning approach, called HoloStain, which converts images of isolated biological cells acquired without staining by holographic microscopy to their virtually stained images. We demonstrate this approach for human sperm cells, as there is a well-established protocol and global standardization for characterizing the morphology of stained human sperm cells for fertility evaluation, but, on the other hand, staining might be cytotoxic and thus is not allowed during human in vitro fertilization (IVF). After a training process, the deep neural network can take images of unseen sperm cells retrieved from holograms acquired without staining and convert them to their stainlike images. We obtained a fivefold recall improvement in the analysis results, demonstrating the advantage of using virtual staining for sperm cell analysis. With the introduction of simple holographic imaging methods in clinical settings, the proposed method has a great potential to become a common practice in human IVF procedures, as well as to significantly simplify and radically change other cell analyses and techniques such as imaging flow cytometry.


Assuntos
Holografia/métodos , Microscopia/métodos , Coloração e Rotulagem/métodos , Algoritmos , Aprendizado Profundo , Citometria de Fluxo , Humanos , Processamento de Imagem Assistida por Computador/métodos , Masculino , Redes Neurais de Computação , Espermatozoides/metabolismo
17.
PLoS Negl Trop Dis ; 14(3): e0008007, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32196491

RESUMO

Investigations into intracellular replication and differentiation of Trypanosoma cruzi within the mammalian host have been restricted by limitations in our ability to detect parasitized cells throughout the course of infection. We have overcome this problem by generating genetically modified parasites that express a bioluminescent/fluorescent fusion protein. By combining in vivo imaging and confocal microscopy, this has enabled us to routinely visualise murine infections at the level of individual host cells. These studies reveal that intracellular parasite replication is an asynchronous process, irrespective of tissue location or disease stage. Furthermore, using TUNEL assays and EdU labelling, we demonstrate that within individual infected cells, replication of both mitochondrial (kDNA) and nuclear genomes is not co-ordinated within the parasite population, and that replicating amastigotes and non-replicating trypomastigotes can co-exist in the same cell. Finally, we report the presence of distinct non-canonical morphological forms of T. cruzi in the mammalian host. These appear to represent transitional forms in the amastigote to trypomastigote differentiation process. Therefore, the intracellular life-cycle of T. cruzi in vivo is more complex than previously realised, with potential implications for our understanding of disease pathogenesis, immune evasion and drug development. Dissecting the mechanisms involved will be an important experimental challenge.


Assuntos
Doença de Chagas/parasitologia , Replicação do DNA , Estágios do Ciclo de Vida , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Modelos Animais de Doenças , Feminino , Genes Reporter , Microscopia Intravital/métodos , Camundongos SCID , Microscopia Confocal/métodos , Coloração e Rotulagem/métodos , Trypanosoma cruzi/genética
19.
Mol Biol (Mosk) ; 54(1): 114-127, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32163395

RESUMO

The factors that affect the labeling of NIH 3T3 murine fibroblasts with Fe3O4-based magnetic nanoparticles (MNPs) were studied using MNPs produced by the gas condensation and solution precipitation methods and MNPs surface-modified with 3-aminopropylsilane or L-lysine. The production method, surface modifications, the particle concentration and size, the state of the cell population, and the method of MNP introduction were found to substantially affect the efficiency of MNP binding by cells. In particular, large MNP clusters may occur in MNP suspensions in DMSO, and their disruption by sonication increased the percent yield of magnetically labeled cells. Static incubation of a cell suspension led to a more efficient labeling as compared with continuous agitation. Cells attached to a plastic support could be labeled to a higher degree than cells in suspension, but required substantially longer incubations with MNPs. MNP centrifugation on cell layers (magnetic spinoculation) significantly increased the rate and efficiency of labeling. The stability of magnetic labeling was shown to depend on the MNP dose during labeling. Electron microscopy studies demonstrated that MNPs were associated with the cell surface after 20-min incubation with cells and were mostly in the cell interior after 4-h incubation. The results of the study may be useful for preparation and application of magnetized cell samples.


Assuntos
Separação Celular/métodos , Nanopartículas de Magnetita/análise , Nanopartículas de Magnetita/química , Coloração e Rotulagem/métodos , Animais , Magnetismo , Camundongos , Células NIH 3T3
20.
PLoS Negl Trop Dis ; 14(3): e0008176, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32214320

RESUMO

BACKGROUND: Schistosome cercariae are the human-infectious stage of the Schistosoma parasite. They are shed by snail intermediate hosts living in freshwater, and penetrate the skin of the human host to develop into schistosomes, resulting in schistosomiasis infection. Water treatment (e.g. filtration or chlorination) is one way of cutting disease transmission; it kills or removes cercariae to provide safe water for people to use for activities such as bathing or laundry as an alternative to infested lakes or rivers. At present, there is no standard method for assessing the effectiveness of water treatment processes on cercariae. Examining cercarial movement under a microscope is the most common method, yet it is subjective and time-consuming. Hence, there is a need to develop and verify accurate, high-throughput assays for quantifying cercarial viability. METHOD: We tested two fluorescence assays for their ability to accurately determine cercarial viability in water samples, using S. mansoni cercariae released from infected snails in the Schistosomiasis Collection at the Natural History Museum, London. These assays consist of dual stains, namely a vital and non-vital dye; fluorescein diacetate (FDA) and Hoechst, and FDA and Propidium Iodide. We also compared the results of the fluorescence assays to the viability determined by microscopy. CONCLUSION: Both fluorescence assays can detect the viability of cercariae to an accuracy of at least 92.2% ± 6.3%. Comparing the assays to microscopy, no statistically significant difference was found between the method's viability results. However, the fluorescence assays are less subjective and less time-consuming than microscopy, and therefore present a promising method for quantifying the viability of schistosome cercariae in water samples.


Assuntos
Cercárias/crescimento & desenvolvimento , Microscopia de Fluorescência/métodos , Carga Parasitária/métodos , Schistosoma mansoni/crescimento & desenvolvimento , Coloração e Rotulagem/métodos , Purificação da Água , Água/parasitologia , Animais , Transmissão de Doença Infecciosa/prevenção & controle , Esquistossomose mansoni/prevenção & controle , Resultado do Tratamento
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